| Summary: | Time courses for the autooxidation of native and mutant sperm whale and pig myoglobins were measured at 37$\sp\circ$C in the presence of catalase and superoxide dismutase. In sperm whale myoglobin, His$\sp{64}$ (E7) was replaced with Gln, Gly, Val, Leu, and Phe; Val$\sp{68}$ (E11) was replaced with Ala, Ile, and Phe; Leu$\sp{29}$ (B10) was replaced with Ala, Val, and Phe. In pig myoglobin, His$\sp{64}$ (E7) was replaced with Val; Val$\sp{68}$ (E11) was replaced with Thr and Ser; Thr$\sp{67}$ (E10) was replaced with Ala, Val, Glu, and Arg; Lys$\sp{45}$ (CD3) was replaced with Ser, Glu, His, and Arg. The observed pseudo first order rate constants varied 5 orders of magnitude, from 44 hr$\sp{-1}$ (H64G) to 0.055 hr$\sp{-1}$ (native) to 0.005 hr$\sp{-1}$ (L29F) at 37$\sp\circ$C, pH 7 in air. The dependence of autooxidation on oxygen and pH were measured for selected proteins. In the native proteins and in most mutants still possessing the distal histidine, autooxidation occurs through a combination of two mechanisms. At high oxygen levels, autooxidation proceeds by direct dissociation of the neutral superoxide radical (HO$\sb2$) from oxymyoglobin, and this process is accelerated by decreasing pH. At low oxygen levels, autooxidation also occurs by a bimolecular reaction between molecular oxygen and a very weakly bound complex between water and ferrous deoxymyoglobin. The neutral side chain of the distal histidine (His$\sp{64}$) inhibits autooxidation by hydrogen bonding to bound oxygen. Replacement of His$\sp{64}$ by amino acids incapable of hydrogen bonding to the bound ligand causes a change in the mechanism of autooxidation and marked increases in the rate of autooxidation. Increasing the polarity of the distal pocket by substitution of Val$\sp{68}$ with Ser and Thr accelerates autooxidation, presumably by facilitating protonation of the Fe(II)-O$\sb2$ complex. Increasing the net anionic charge at the protein surface in the vicinity of the heme group also enhances the rate of autooxidation. Decreasing the volume ...
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